Emerging Approaches to Understanding Microvascular Endothelial Heterogeneity: A Roadmap for Developing Anti-Inflammatory Therapeutics

Abstract

The endothelium is the inner layer of all blood vessels and it regulates hemostasis. It also plays an active role in the regulation of the systemic inflammatory response. Systemic inflammatory disease often results in alterations in vascular endothelium barrier function, increased permeability, excessive leukocyte trafficking, and reactive oxygen species production, leading to organ damage. Therapeutics targeting endothelium inflammation are urgently needed, but strong concerns regarding the level of phenotypic heterogeneity of microvascular endothelial cells between different organs and species have been expressed. Microvascular endothelial cell heterogeneity in different organs and organ-specific variations in endothelial cell structure and function are regulated by intrinsic signals that are differentially expressed across organs and species; a result of this is that neutrophil recruitment to discrete organs may be regulated differently. In this review, we will discuss the morphological and functional variations in differently originated microvascular endothelia and discuss how these variances affect systemic function in response to inflammation. We will review emerging in vivo and in vitro models and techniques, including microphysiological devices, proteomics, and RNA sequencing used to study the cellular and molecular heterogeneity of endothelia from different organs. A better understanding of microvascular endothelial cell heterogeneity will provide a roadmap for developing novel therapeutics to target the endothelium.

Keywords: bMFA; endothelial barrier permeability; heterogeneity; inflammation; leukocytes; microphysiological systems; microvascular endothelial cells; protein kinase Cδ; sepsis; transmigration.

Figure 1

Endothelial cell activation in sepsis: increased barrier permeability and neutrophil migration: (A) Under normal conditions, the vascular endothelium is covered by the glycocalyx to form a tight barrier that regulates barrier permeability, neutrophil migration, and anti-inflammatory defenses. (B) During sepsis, PAMPS and DAMPS activate neutrophils and endothelial cells to produce cytokines and chemoattractants, which activate neutrophils to display surface molecules that interact with adhesion molecules expressed by activated endothelium. The rolling step involves interactions of E/P-selectin and their ligand (e.g., PSGL-1) on neutrophils, which slows down the neutrophil. The next step, firm adhesion, is mediated by adhesion molecules of endothelium, including ICAM-1, ICAM-2, and VCAM-1, and their neutrophil ligands, β₂ integrins. In response to chemoattractants, adhered neutrophils migrate through endothelial junctions, involving PECAM-1 and JAMs. Activated neutrophils release cytokines, ROS, and proteases, or undergo NETs formation. During sepsis, the glycocalyx is degraded, endothelial cell tight junctions are damaged, and there is increased endothelial cell apoptosis, leading to damaged barrier function and increased permeability.

Figure 2

The biomimetic microfluidic assay (bMFA) mimics a physiologically relevant microvascular environment, which is used to study neutrophil-endothelial cell interactions: (A) The bMFA includes vascular channels and tissue compartment, which are connected through a 3 μm barrier region (scale bar 500 μm). (B) Microvascular network maps obtained in vivo are reproduced on PDMS to assemble the bMFA (scale bar 1 cm). ((A) Reproduced with permission from reference [127], https://pubs.acs.org/doi/10.1021/ac5018716, accessed on 25 May 2021. further permissions related to the material excerpted should be directed to the American Chemical Society. (B) Reproduced with permission from reference [140]).

Figure 3

Endothelial cells form a complete lumen in the bMFA: (A) phase contrast images show endothelial cells are lined up in the direction of flow (scale bar is 100 µm). (B) Confocal micrograph of endothelial cells showing 3D lumen formation in the vascular channel; F-actin is labeled in green and nuclei is labeled in red. (Reproduced with permission from reference [120]).

Figure 4

In vitro neutrophil migration in response to inflammation: regulation by PKCδ: (A) TNF-α treatment significantly increased human neutrophil adhesion to human pulmonary microvascular endothelial cells, which was inhibited following treatment with the PKCδ inhibitor. Neutrophil adhesion occurred preferentially at low shear rates and near bifurcations in the bMFA. (B) In response to fMLP, human neutrophil migration across TNFα-activated endothelial cells was significantly increased compared to untreated cells. Treatment with the PKCδ inhibitor reduced migration to untreated levels. (C) TNFα treatment increased the expression of the adhesion molecules, E-selectin, ICAM-1, and VCAM-1, but not JAM-C, indicating selective regulation. PKCδ inhibitor treatment significantly decreased E-selectin, ICAM-1, and VCAM-1, but not JAM-C expression. (Reproduced with permission from reference [47]) (n = 4 for panel A and B, n = 3 for panel C, data is presented as Mean ± SEM, one-way ANOVA, ** p < 0.01, and *** p < 0.001).